Objective: To determine the effect of repeated freezing and thawing, and storage temperature, on the assay results of commonly measured reproductive hormones, and sex hormone-binding globulin (SHBG) in human serum. Design: Prospective laboratory study. Setting: Academic medical center. Patient(s): Four men and three pregnant women in the third trimester. Intervention(s): Pooled serum from men and pregnant females were frozen at either -20°C or -70°C. Aliquots were then subjected to repeat freeze/thaw cycles, from 1 to 10 times, and assays were performed after the final freeze/thaw cycle. Main Outcome Measure(s): Assay results for eight hormones (FSH, LH, PRL, androstenedione (A), 17α-hydroxyprogesterone, P, insulin, and SHBG, as a function of the number of freeze/thaw cycles and storage temperature. Result(s): Only SHBG in male serum at -20°C and P in pregnant serum at -70°C showed statistically significant decreases in assay results with repeated freeze/thaw cycles (3.3% and 1.1% per cycle, respectively). All other analytes did not show significant changes as a function of freeze/thaw cycles or storage temperature. Conclusion(s): There is no consistent or predictable alteration in the results of SHBG, or the glycoprotein and steroid hormones evaluated, as a function of repeated freeze/thaw or the storage temperature of human serum. © 2001 by American Society for Reproductive Medicine.